CN107937484A - Liver regeneration correlation lncRNA and its screening technique, inhibitor and application - Google Patents
Liver regeneration correlation lncRNA and its screening technique, inhibitor and application Download PDFInfo
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Abstract
The present invention relates to the method for screening liver regeneration correlation long-chain non-coding RNA (long non coding RNA, lncRNA) and inhibitor and its application of lncRNA TCONS_00027980.Specifically construct 2/3 hepatectomy model of rat, go out the differential expression lncRNA in liver regeneration using 2.0 cDNA microarrays of rat lncRNA array v, the function of lncRNA is predicted with association presumption method, the relevant lncRNA of liver regeneration is filtered out, including lncRNA TCONS_00027980, its transcript nucleotide sequence is as shown in SEQ ID NO.1, according to the sequence, its inhibitor is designed and synthesized, inhibitor provided by the invention can suppress the lncRNA TCONS_00027980 expression of the BRL 3A cells of in vitro culture, promote the cell Proliferation.The present invention can provide new drug target for prognosis of orthotopic liver transplantation auxiliary treatment, have a good application prospect and theory value.
Description
Technical field
The invention belongs to biomedicine technical field, and in particular to a kind of liver regeneration correlation long-chain non-coding RNA and its suppression
Preparation, and the application in the preparation for adjusting hepatocyte growth is prepared.
Background technology
Liver is one of most important organ in humans and animals body, carries different physiological roles.Liver participate in carbohydrate, lipid,
The metabolic process of the materials such as protein.The factors such as liver trauma, operation, poisonous substance, infection cause repairing again for liver cell after hepatic injury
It is multiple, i.e., it is liver regeneration by the process of the physiological activities such as cell Proliferation and differentiation recovery liver original structure and function.Liver regeneration relates to
And multiple physiological and biochemical procedures such as cell-stimulating, cell de-differentiation, cell Proliferation, Apoptosis and institutional framework reconstruction, including
Many factors and liver structure, function, disease generation etc. including microRNAs are closely related.Therefore, the molecule of liver regeneration is illustrated
Mechanism, reparation, liver transfer operation, liver disease drug exploitation, liver disease and prevention to liver trauma etc. is significant.
Long-chain non-coding RNA (long non-coding RNA, lncRNA) refers to that few nucleotide is more than the non-volume of 200nt
Code rna transcription sheet.LncRNA species is enriched, and function is complicated, and positioned at different subcellular fraction core regions, sequence is protected between different plant species
Keeping property is low, specific with the stage of development in a organized way, while is the non-coding RNA molecule with tachytelic evolution potentiality again.lncRNA
It is transcribed from gene and non-genomic region, including the upstream region of gene, intergenic region, gene intron region etc., mostly
Several lncRNA is determined to be the key regulator in transcription and translation, has important shadow in the performance of cell normal function
Ring.For example participate in adjusting, intracellular matter transport, the formation of nucleus substructure, stem cell after chromatin remodeling, transcription and transcription
Versatility, reprogramming of somatic cells, growth adjustment and disease generation etc..When playing these functions, lncRNA passes through different works
The adjusting to gene expression, including cis adjusting and trans regulative mode are realized with approach and mode.In addition, lncRNA can be borrowed
Protein is helped with both different approaches of microRNA networks to play specific effect.
Though lncRNA, without the ability of coding protein, there are some researches prove it participates in cell Proliferation, cell growth, thin
A variety of physiological activities such as born of the same parents' differentiation, apoptosis.And have proven to a variety of diseases especially cancer such as liver cancer, breast cancer, oophoroma, stomach
Cancer etc. is directed to the unconventionality expression of lncRNA.Drug research new direction using RNA as target spot has more and more been paid attention to,
The methods of by designing small-molecule drug, antisense oligonucleotides, ribozyme, acts on RNA, this should be that future drugs are researched and developed
New direction.
The content of the invention
It is an object of the invention to provide a kind of long-chain non-coding RNA (long non-coding RNA, lncRNA) TCONS_
00027980 application process, specifically adjusts hepatocyte growth using lncRNA TCONS_00027980 for target spot.
The method of one kind screening liver regeneration correlation long-chain non-coding RNA (long non-coding RNA, lncRNA), institute
The method of stating includes：
1) liver regeneration rat model is built；
2) 2.0 cDNA microarrays of rat lncRNA Array v are used, obtain out differential expression in liver regeneration
lncRNA；
3) by associating presumption method prediction steps 2) in differential expression lncRNA function, filtered out liver regeneration correlation
LncRNA.
In an embodiment according to the present invention, rat described in step 1) is adult healthy male Sprague-
Dawley rats, the liver regeneration rat model are that rat liver is cut off 2/3.
In an embodiment according to the present invention, the lncRNA of differential expression described in step 2) is monitoring hepatectomy
After processing during 0,2 and 6h differential expression lncRNA.
In an embodiment according to the present invention, the association presumption method described in step 3) is by by every
The expression value of lncRNA carries out coexpression analysis with the expression value of all differences expressing gene, and it is corresponding common to find every lncRNA
Target gene set is expressed, the enrichment analysis of GO and KEGG functions is carried out to the target gene set of each lncRNA, to predict every
The function of lncRNA.
In an embodiment according to the present invention, the corresponding coexpression target gene of every lncRNA be with it is specific
LncRNA expression value related coefficients absolute value is more than 0.8, and the p value of correlation test is less than 0.05 gene.
Present invention also offers a kind of liver regeneration correlation long-chain non-coding RNA (long non-coding RNA,
LncRNA), the lncRNA screens to obtain by the above method；Preferably, the liver regeneration correlation long-chain non-coding RNA
For lncRNA TCONS_00027980.
Invention further provides a kind of inhibitor for being used to adjust hepatocyte growth, the inhibitor is to be used to adjust
The DNA or RNA of lncRNA TCONS_00027980 transcript nucleotide sequence expressions；Preferably, the inhibitor is base
In the siRNA, the lncRNA TCONS_00027980 of the design of lncRNA TCONS_00027980 transcripts nucleotide sequence
Transcript nucleotides sequence is classified as SEQ ID NO.1.
In an embodiment according to the present invention, the inhibitor is the one or more in 6 kinds of siRNA；
1)siRNA-1：SEQ ID NO：6atgatgtgggatgtacaag
2)siRNA-2：SEQ ID NO：7tgcttgatgatgtaggtcc
3)siRNA-3：SEQ ID NO：8tcattgttgatttgtcagc
4)siRNA-4：SEQ ID NO：9agccatgatgtgggatgtac
5)siRNA-5：SEQ ID NO：10cctccttccttcatgtggac
6)siRNA-6：SEQ ID NO：11ccctcctcgtgtgattgcag.
Present invention also offers a kind of preparation for being used to adjust hepatocyte growth, the preparation includes such as claim 8 or 9
The inhibitor.Said preparation can be medical medicine composition or reagent for experimental purposes, such as add medicine
Acceptable auxiliary material on, or solvent in experimental technique etc..
Compared with prior art, the present invention has at least the following advantages：
The present invention successfully filter out lncRNA TCONS_00027980, its can be used as adjusting hepatocyte growth target spot,
The inhibitor of lncRNA TCONS_00027980 can be further used for preparing the system for promoting hepatocyte growth, and the present invention can be liver
Transplant prognosis auxiliary treatment and strong biology tool is provided, there is important clinical meaning and application prospect.
Brief description of the drawings
Fig. 1 is shadow of the inhibitor to BRL-3A cell-proliferation activities that lncRNA TCONS_00027980 are detected with mtt assay
Ring；
Fig. 2A, Fig. 2 B and Fig. 2 C illustrate the inhibitor of lncRNA TCONS_00027980 to the BRL-3A cell cycles
Influence；Wherein Fig. 2A is the flow cytometer result of control group, the flow cytometer that Fig. 2 B are test group as a result, Fig. 2 C are control
The column diagram that the cell cycle of group and test group contrasts.
Embodiment
The invention will be further described with reference to the accompanying drawings and examples, and following embodiments are descriptive, are not
Limited, it is impossible to protection scope of the present invention is limited with this.
Embodiment 1：Liver Regeneration of Rat model prepares and materials
Experimental rat is adult healthy male Sprague-Dawley rat, 230 ± 20g of weight, by He'nan Normal University
Experimental Animal Center provides.Raising temperature is 21 ± 2 DEG C, and relative humidity is 60 ± 10%, light application time 12h/d (8:00-20:
00), free water, ingest.114 above-mentioned rats are taken, are randomly divided into 19 groups, every group 6.Wherein, 1 group of normal control (0h),
It is 9 groups each that 2/3 hepatectomy (partial hepatectomy, PH) compares (sham operation, SO) with sham-operation.PH groups are pressed
Higgins and Anderson methods carry out, and SO groups are not in addition to lobe of the liver is cut off, other same PH.After operation during 2,6h, right lobe of liver is taken to put
In tissue storage reagent (such as RNALater), saved backup in -20 DEG C.
Embodiment 2：The high-flux sequence of lncRNAs
Take the appropriate above-mentioned hepatic tissue for being stored in -20 DEG C to be placed in the mortar equipped with liquid nitrogen to grind, by total RNA extraction reagent
Box (Ambion, the U.S.) operating guidance extracts and purifying total serum IgE s.Total serum IgE is detected with agarose gel electrophoresis (180V, 0.5h)
Quality, rRNA: 18S rRNA of 28S are about 2: 1, measure OD respectively260And OD280, in OD260/OD280>=2.0, it is considered as RNA conjunctions
Lattice.The qualitative and quantitative analysis of miRNAs is carried out by Shanghai Bai Hao biotech companies, with single-ended Solexa microRNA-Seq
High-flux sequence method, is examined from the Regenerating Liver of Rat at 3 time points such as 0,2 and 6h of experimental group (PH) and sham-operation group (SO)
Survey.
Embodiment 3：The screening of lncRNA and mRNA gene expression abundances and differential expression lncRNA
Expression quantity of the mRNA in three samples is analyzed using cuffdiff softwares, using classical RPKM
(Reads Per Kilobases per Million reads) formula calculation expression amount.All turns will rebuild from sample
Record this (or seeing locus) integrally carries out Differential expression analysis, as a result the preference without molecule type.For abiology repeating sample
Project design, we using DESeq carry out Differential expression analysis, and by expression value fold differences for log2 (FC) absolute value it is big
Liver regeneration correlation lncRNA is defined as in genes of the 1 and p-value less than 0.05 or transcript.High-flux sequence the result shows that,
The 2h after PH, detects 911 lncRNA, wherein 522 related to liver regeneration；The 6h after PH, detects 908 lncRNA,
Wherein 680 (table 1) related to liver regeneration.
LncRNA transcripts expression variable number statistics in 1 Liver Regeneration of Rat of table
Embodiment 4：The function prediction of lncRNA
, can be by the expression of the expression value of every lncRNA and all differences expressing gene using 0,2 and 6h, tri- samples
Value carries out coexpression analysis, and determining every lncRNA, there are coexpression relation with which difference expression gene.In concrete analysis,
It is by coexpression contextual definition：Specific lncRNA is more than 0.8 with specific gene expression value related coefficient absolute value, and correlation is examined
The p value tested is less than 0.05.By this definition, find the corresponding coexpression target gene set of every lncRNA, and using GO and
KEGG carries out target gene function enrichment analysis, and the function prediction result to lncRNA is used as using this result.To each
The target gene set of lncRNA carries out the enrichment analysis of GO and KEGG functions, to predict the function of every lncRNA.
Embodiment 5：Real-time fluorescence quantitative PCR detects
LncRNA sequencing results need the reliability of further verification result.To select differential expression in experiment
LncRNA, is changed with the expression of RT-PCR detections different time points after rat hepatectomy.Experiment utilizes Oligo 6 and Primer
The primer (table 2) of 5.0 Software for Design of Premier detection lncRNA and internal reference.Primer is synthesized by Shanghai Bio-engineering Corporation.
Total serum IgE in rats'liver is extracted by Trigol kit specifications.According to the operation in AMV Reverse Transcriptase kits (Promega, the U.S.)
Illustrate that carrying out reverse transcription obtains cDNA.Then by the use of the cDNA as template, and screened the optimal reaction temperature of PCR, the time and
Template amount.Then quantitative fluorescent PCR is carried out, the condition of quantitative fluorescent PCR is：95 DEG C of 2min, 95 DEG C of 15sec, 60 DEG C
20sec, 72 DEG C of 20sec, 40 circulations.Each sample repeats detection three times, and the data obtained is with 2-ΔΔCtMethod is made at relative quantification
Reason.The lncRNA being consistent with high throughput testing result is obtained, and using them as candidate lncRNA.
2 real-time fluorescence quantitative PCR primer sequence of table
Real-time fluorescence quantitative PCR detects expressions of the lncRNA TCONS_00027980 in 0,2 and 6h of liver regeneration.
The result shows that two kinds of expression trend for differently detecting lncRNA are consistent, data are credible.
Embodiment 6：The design and synthesis of the inhibitor of lncRNA TCONS_00027980
Transcript nucleotide sequence of the present embodiment according to lncRNA TCONS_00027980, has designed and synthesized suppression
Agent (table 3).
The inhibitor nucleotide sequence of 3 lncRNATCONS_00027980 of table
Embodiment 7：Rat hepatocytes culture
Rat normal liver cell BRL-3A used in experiment is purchased from Shanghai Inst. of Life Science, CAS cellular resources
Center, culture medium are the DMEM high glucose mediums containing 10% hyclone (FBS), cell culture in 37 DEG C, saturated humidity, 5%
CO2Incubator in.Growth period cell of taking the logarithm is passed on, and 5 × 104A cell/bottle.
Embodiment 8：Mtt assay detects hepatocyte growth
Take the logarithm the BRL-3A cells in growth period, 0.25% pancreatin (Invitrogen, the U.S.) digestion, by 1ml/ holes, containing 5
×104The cell suspension inoculation of a/ml cells is in 24 porocyte culture plates, in 37 DEG C, 5%CO2, cultivate under saturated humidity, carefully
Born of the same parents' density is transfected when reaching 30-50%, with reference to riboFECTTMCP (sharp rich, China) transfection reagent operation instruction is turned
Dye.3 multiple holes of each experimental setup, while blank control and negative control are set.Experiment is repeated 3 times.
10 μ l/ hole MTT (Geneview, the U.S.) are added in 96 orifice plates containing cell and culture medium, make its ultimate density
Up to 0.5g/L, in 37 DEG C of lucifuge culture 4h, after thoroughly discarding nutrient solution, added per hole 100 μ l dimethyl sulfoxide (DMSO)s (Geneview,
The U.S.), 10min is gently shaken, fully dissolves first a ceremonial jade-ladle, used in libation crystal.Finally, with microplate reader (Biotek, the U.S.) at 490nm wavelength
Detect the light absorption value in each hole.Experiment is repeated 3 times, and each experimental group sets 3 multiple holes.Testing result is as shown in Figure 1, concentration is
The inhibitor of 100nM lncRNA TCONS_00027980, after transfectional cell during 48,72 and 96h experimental group absorbance with it is right
It is obvious according to group difference.
Embodiment 9：Cell cycle is detected
, will for influences of the detection lncRNA TCONS_00027980 to the cell cycle of rat normal liver cell BRL-3A
Cell inoculation is in 24 porocyte culture plates, and 1 × 105A cells/well, when cell grows to 50~60% degree of converging, adds fat
Plastid 2000 (Invitrogen, the U.S.) is compareed with analogies, analogies respectively, the control of inhibitor, inhibitor, inhibitor transfection
Concentration is 100nM/ holes.In 5%CO2, cultivate 48h in 37 DEG C of incubators after collect cell.It is fixed thin in -20 DEG C with 70% ethanol
Born of the same parents stay overnight, with PBS wash and 200 mesh sieve net filtration cells after, then with propidium iodide dye liquor (50 μ g/mL PI, 100 μ g/mL
RNase A) room temperature lucifuge dyeing 30min, with the Flow cytometry cell cycle.Flow cytometry the results show (figure
2A, Fig. 2 B and Fig. 2 C), the flow cytomery cell cycle the result shows that, transfect lncRNA TCONS_00027980 inhibitor
48h afterwards, experimental group (S+G2M%) cell proliferation rate are (36.4 ± 1.833) %, and negative control group cell proliferation rate is (23.92
± 1.931) %, both S+G2M cell numbers significant differences (p ＜ 0.05) show to reduce lncRNA TCONS_002798027980
Expression increases ratio of the BRL-3A cells in S+G2M.Therefore increasings of the lncRNA TCONS_00027980 to BRL-3A cells
Grow with obvious inhibitory action.
More than, it is merely preferred embodiments of the present invention, but the protection domain invented is not limited thereto, and is replaced,
It should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of claims
Subject to enclosing.
Sequence table
<110>He'nan Normal University
<120>Liver regeneration correlation lncRNA and its screening technique, inhibitor and application
<130> EY01PT011702323
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<170> SIPOSequenceListing 1.0
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aaaatctcat cacaggtcag tcaatagata tttctaaaca atccttgtac gtttctcggc 120
caatcttttt cttctatttt cagagatctc ttattttcat tcagaaaaaa aaaatctcat 180
cacaggtcag tcaatagata tttctaaaca atccttgtac atcccacatc atggctaacc 240
tagaagaaaa taaagtatca gacagtaagg gcttcaattt gaaatcagag gaaaacaaaa 300
ctaacaaaat cccagcaagc aagcacttgc tgaagtgtat ctgtgggaga ggaccgagag 360
cctaaggaaa agtgagcgtc cacaccagtt ttctaggaac ccattctgtt tacacctgct 420
acattggtgt aattgttatt attaatccct caaccttcaa aactgtaccg gtttggatga 480
taaaccataa agttacctta atgatggctc atctatagaa gtggattcaa caaagactat 540
aaccatagca actaggaacc atgctgatgg gcgctttgtt tgcacagatg acctcacact 600
ccgagtgtat tttagcttgg atgagacaat tccttctatt gccggtgaga tttgtaaagg 660
ctgggcttgt cctgttggtt aatctgatgc aaagactctt ctctgtgttc atattctgat 720
ctctgggata ggcttagact actgctaaga catgccgggt cttttgtcta tgggaaacag 780
gtcaagatca gccctaagga atgggcaaaa agtgaacggt agtcacttca gtgtagtcca 840
catgaaggaa ggaggcagga ggtctcacca gctcagcttg acagcctggt ctgagcatcc 900
actccatgct gggtgctcag cctttagtca gtgagacaga aggaattgca tgagcaacaa 960
cagaatagtc aagcactaag aaaagcttgg cctgcgatca atttggtgtt ggtggaagtg 1020
tgcattatac atcagtggga ggcaggagac atggctgtcg atgctggcag aggctaaatg 1080
atggaggacc tacatcatca agcattatgg gtttcctctg gaattgaaac ggcatctcca 1140
atgtcatccc atcctggggc ctctccttgt tctgacagtt acattttgca gcaagatgac 1200
ctggttttca ggatcttact gatctctcag aacctctgtg ctccccacct ggggctctgt 1260
ccatgtcttg atcacattat cagatgtagc agatctgatg acacagttcc tagggatcca 1320
ggacaaagag taccagcagc aactgccagc cttgggcact cgtcctcgtg tttgtggtca 1380
cctctagacg ctacagaaga actgatgcag caagaacaat cgattgatgg gggtcaagtt 1440
ggggtttcca cagcaattgc tgacaatgcc gagctgacat agaaggcgct gtctactctt 1500
ctggggcact tagatggtat aaaaggccac ttaacccagt gtgcagttgc agccttgagc 1560
tgcgaatctt gaactccctg ccattctgat gtacagcatg taaaaagcac gtgattatgt 1620
ttatcctggg agtgcgcgct cttggccaca gcagcagatc agtctggaat aagcaaatag 1680
aatcacccca aaaggcaaac tgcaaaagct atctgcaatc acacgaggag ggcatgccaa 1740
cagagattct tttcaaagaa gagaattaca ggaaagaaat agatgactaa gaacttttaa 1800
aaaatcaagt gcagacgttt gttttcccat ttgctagacg tgagggctaa acatgatact 1860
gtctctaggg ttgatttgct gacaaatcaa caatgatgtg agggagtgtg gccagccttc 1920
tttttcccct gaggacggcg gagatgccaa gagatggagg ccatcttgtc taaaatagga 1980
ccgtgaggtt actggctgag tttggtctat cctaggtcat gtggataccc agttcacctt 2040
ggatatccat ccaaaatgcc taagtgagta tatggtgatg gagtttaaat tgtgaccctc 2100
cctggagaga agactccaga ccgttcacgt ttcctggctg gtcacaccag cactattctg 2160
cctgatgact tgacaaactt ggtccagaat ctacttcctt tgtacaggaa ttcaggctct 2220
atgaaatcta tcagccaagc cacaatggct ttggtttttc aagacaagct ctctctgtgt 2280
agccccgaat gtcctggaac tccctctgaa tttgaactca gagatccact tgcctctgcc 2340
tccaggctgc tgggactaaa ggtgtccacc tccacttcca gaagacaaac ttcttgccag 2400
ggaggctgac atacattgtg atgtaaactc ccttgactgg tccagtttgg tagccattgg 2460
gctccattac ctatctgtag gcagctcttt gagaaacact gacatattca tggctgtggc 2520
ttgggccatc ttcacaggcc agaaagggct tcgtttatca gtcagtcaac atgcagagag 2580
aacagtttgt tttggaacaa ttcaagtctg taaaaagatc caatagtaca aagacctcct 2640
gggtgccttc cacctgggtc tcttttgaat tctgcctatg gtgtacctgc tactaagggg 2700
tcaattcatg aactagattt cctatctgtt tcttcatcag agcaccgtgc cctcaacctc 2760
atgaggacct cattacacat gatcacaggg ttttccctat acagcctcat agcaatctgg 2820
gtcatatatt tttggcaaaa aaatgttaga aaagtgattc agtgcttctt tactgctccc 2880
ttaccaagtg gcccttgtga atttgttcca atgcttgtgg tagtgggttc gattgcttga 2940
ttgagatggg gtttgcccct gtttttttct ttttattaag gttgcttttt ttaaaaaaat 3000
gtgattaata ttatataaat atttcattac ccagtctaca accgtgtgtg tgtgtgtgtg 3060
tgtgtgtgtg tgtgtgtgtg tgtgtgtgta tgtgtgtgta tgtgtgtgta tgtgtgtgtg 3120
tgtgaaggct ggagctccct gttgacagtc tttctcaatt actctccctt gatttctaga 3180
aacagggtct ctcactgaag cgggacctcc cagttcggtt agactggtaa gcctctgggt 3240
cctcttgtcc atgccctggg ccttcccagc tctgggtcta gaaatgtctg cttccctact 3300
tggtttttca catgggtgct gggggtcaag tgaaggtctt cacattcagg gggcaaatcc 3360
cccagtcccc acttgtttgc tttaatatca actgatgctt cttgctggag tttttttgcc 3420
aagatgttta ccaaatggta attttctaat tgtgtcactc cctgtatatt taacaactgg 3480
tattcctatg aaagcacaca ctgtctcatc tccattttaa aactcactta taagccattc 3540
actagttaat gggttataat cctttattgc cactttattg atacttaagt tgatctacat 3600
tatgacagag tcccaagcat gtgacatccc acatcatggc taacctagaa gaaaataaag 3660
tatcagacag taagggcttc aatttgaaat cagaggaaaa caaaactaac aaaatcccag 3720
caagcaagca cttgctgaag tgtatctgtg ggagaggacc gagagcctaa ggaaaagtga 3780
gcgtccacac cagttttcta ggaacccatt ctgtttacac ctgctacatt ggtgtaattg 3840
ttattattaa tccctcaacc ttcaaaactg taccggtttg gatgataaac cataaagtta 3900
ccttaatgat ggctcatcta tagaagtgga ttcaacaaag actataacca tagcaactag 3960
gaaccatgct gatgggcgct ttgtttgcac agatgacctc acactccgag tgtattttag 4020
cttggatgag acaattcctt ctattgccgg tgagatttgt aaaggctggg cttgtcctgt 4080
tggttaatct gatgcaaaga ctcttctctg tgttcatatt ctgatctctg ggataggctt 4140
agactactgc taagacatgc cgggtctttt gtctatggga aacaggtcaa gatcagccct 4200
aaggaatggg caaaaagtga acggtagtca cttcagtgta gtccacatga aggaaggagg 4260
caggaggtct caccagctca gcttgacagc ctggtctgag catccactcc atgctgggtg 4320
ctcagccttt agtcagtgag acagaaggaa ttgcatgagc aacaacagaa tagtcaagca 4380
ctaagaaaag cttggcctgc gatcaatttg gtgttggtgg aagtgtgcat tatacatcag 4440
tgggaggcag gagacatggc tgtcgatgct ggcagaggct aaatgatgga ggacctacat 4500
catcaagcat tatgggtttc ctctggaatt gaaacggcat ctccaatgtc atcccatcct 4560
ggggcctctc cttgttctga cagttacatt ttgcagcaag atgacctggt tttcaggatc 4620
ttactgatct ctcagaacct ctgtgctccc cacctggggc tctgtccatg tcttgatcac 4680
attatcagat gtagcagatc tgatgacaca gttcctaggg atccaggaca aagagtacca 4740
gcagcaactg ccagccttgg gcactcgtcc tcgtgtttgt ggtcacctct agacgctaca 4800
gaagaactga tgcagcaaga acaatcgatt gatgggggtc aagttggggt ttccacagca 4860
attgctgaca atgccgagct gacatagaag gcgctgtcta ctcttctggg gcacttagat 4920
ggtataaaag gccacttaac ccagtgtgca gttgcagcct tgagctgcga atcttgaact 4980
ccctgccatt ctgatgtaca gcatgtaaaa agcacgtgat tatgtttatc ctgggagtgc 5040
gcgctcttgg ccacagcagc agatcagtct ggaataagca aatagaatca ccccaaaagg 5100
caaactgcaa aagctatctg caatcacacg aggagggcat gccaacagag attcttttca 5160
aagaagagaa ttacaggaaa gaaatagatg actaagaact tttaaaaaat caagtgcaga 5220
cgtttgtttt cccatttgct agacgtgagg gctaaacatg atactgtctc tagggttgat 5280
ttgctgacaa atcaacaatg atgtgaggga gtgtggccag ccttcttttt cccctgagga 5340
cggcggagat gccaagagat ggaggccatc ttgtctaaaa taggaccgtg aggttactgg 5400
ctgagtttgg tctatcctag gtcatgtgga tacccagttc accttggata tccatccaaa 5460
atgcctaagt gagtatatgg tgatggagtt taaattgtga ccctccctgg agagaagact 5520
ccagaccgtt cacgtttcct ggctggtcac accagcacta ttctgcctga tgacttgaca 5580
aacttggtcc agaatctact tcctttgtac aggaattcag gctctatgaa atctatcagc 5640
caagccacaa tggctttggt ttttcaagac aagctctctc tgtgtagccc cgaatgtcct 5700
ggaactccct ctgaatttga actcagagat ccacttgcct ctgcctccag gctgctggga 5760
ctaaaggtgt ccacctccac ttccagaaga caaacttctt gccagggagg ctgacataca 5820
ttgtgatgta aactcccttg actggtccag tttggtagcc attgggctcc attacctatc 5880
tgtaggcagc tctttgagaa acactgacat attcatggct gtggcttggg ccatcttcac 5940
aggccagaaa gggcttcgtt tatcagtcag tcaacatgca gagagaacag tttgttttgg 6000
aacaattcaa gtctgtaaaa agatccaata gtacaaagac ctcctgggtg ccttccacct 6060
gggtctcttt tgaattctgc ctatggtgta cctgctacta aggggtcaat tcatgaacta 6120
gatttcctat ctgtttcttc atcagagcac cgtgccctca acctcatgag gacctcatta 6180
cacatgatca cagggttttc cctatacagc ctcatagcaa tctgggtcat atatttttgg 6240
caaaaaaatg ttagaaaagt gattcagtgc ttctttactg ctcccttacc aagtggccct 6300
tgtgaatttg ttccaatgct tgtggtagtg ggttcgattg cttgattgag atggggtttg 6360
cccctgtttt tttcttttta ttaaggttgc tttttttaaa aaaatgtgat taatattata 6420
taaatatttc attacccagt ctacaaccgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 6480
gtgtgtgtgt gtgtatgtgt gtgtatgtgt gtgtatgtgt gtgtgtgtga aggctggagc 6540
tccctgttga cagtctttct caattactct cccttgattt ctagaaacag ggtctctcac 6600
tgaagcggga cctcccagtt cggttagact ggtaagcctc tgggtcctct tgtccatgcc 6660
ctgggccttc ccagctctgg gtctagaaat gtctgcttcc ctacttggtt tttcacatgg 6720
gtgctggggg tcaagtgaag gtcttcacat tcagggggca aatcccccag tccccacttg 6780
tttgctttaa tatcaactga tgcttcttgc tggagttttt ttgccaagat gtttaccaaa 6840
tggtaatttt ctaattgtgt cactccctgt atatttaaca actggtattc ctatgaaagc 6900
acacactgtc tcatctccat tttaaaactc acttataagc cattcactag ttaatgggtt 6960
ataatccttt attgccactt tattgatact taagttgatc tacattatga cagagtccca 7020
agcatgtgac 7030
<210> 2
<211> 25
<212> DNA
<213> Human artificial
<400> 2
tccctataca gcctcatagc aatct 25
<210> 3
<211> 23
<212> DNA
<213> human artificial
<400> 3
caagcaatcg aacccactac cac 23
<210> 4
<211> 17
<212> DNA
<213> human artificial
<400> 4
ctcgcttcgg cagcaca 17
<210> 5
<211> 20
<212> DNA
<213> Human artificial
<400> 5
aacgcttcac gaatttgcgt 20
<210> 6
<211> 12
<212> DNA
<213> Human artificial
<400> 6
atgatgtggg at 12
<210> 7
<211> 13
<212> DNA
<213> Human artificial
<400> 7
tgcttgatga tgt 13
<210> 8
<211> 13
<212> DNA
<213> Human artificial
<400> 8
tcattgttga ttt 13
<210> 9
<211> 11
<212> DNA
<213> Human artificial
<400> 9
agccatgatg t 11
<210> 10
<211> 13
<212> DNA
<213> Human artificial
<400> 10
cctccttcct tca 13
<210> 11
<211> 12
<212> DNA
<213> Human artificial
<400> 11
ccctcctcgt gt 12
Claims (10)
1. the method for one kind screening liver regeneration correlation long-chain non-coding RNA (long non-coding RNA, lncRNA), it is special
Sign is, the described method includes：
1) liver regeneration rat model is built；
2) 2.0 cDNA microarrays of rat lncRNA Array v are used, obtain out the lncRNA of differential expression in liver regeneration；
3) by associating presumption method prediction steps 2) in differential expression lncRNA function, it is relevant to have filtered out liver regeneration
lncRNA。
2. the method as described in claim 1, it is characterised in that rat described in step 1) is adult healthy male Sprague-
Dawley rats, the liver regeneration rat model are that rat liver is cut off 2/3.
3. the method as described in claim 1, it is characterised in that the lncRNA of differential expression described in step 2) cuts for monitoring liver
The lncRNA of differential expression during except after processing 0,2 and 6h.
4. the method as described in claim 1, it is characterised in that the association presumption method described in step 3) is by by every
The expression value of lncRNA carries out coexpression analysis with the expression value of all differences expressing gene, and it is corresponding common to find every lncRNA
Target gene set is expressed, the enrichment analysis of GO and KEGG functions is carried out to the target gene set of each lncRNA, to predict every
The function of lncRNA.
5. method as claimed in claim 4, it is characterised in that the corresponding coexpression target gene of every lncRNA is and spy
LncRNA expression value related coefficients absolute value is determined more than 0.8, and the p value of correlation test is less than 0.05 gene.
6. a kind of liver regeneration correlation long-chain non-coding RNA (1ong non-coding RNA, lncRNA), it is characterised in that described
LncRNA is obtained by the screening of such as any one of claim 1~6；Preferably, the non-volume of liver regeneration correlation long-chain
Code RNA is lncRNA TCONS_00027980.
7. a kind of inhibitor for being used to adjust hepatocyte growth, it is characterised in that the inhibitor is to be used to adjust lncRNA
The DNA or RNA of TCONS_00027980 transcript nucleotide sequence expressions.
8. inhibitor as claimed in claim 7, it is characterised in that the inhibitor is to be based on lncRNA TCONS_
The siRNA of 00027980 transcript nucleotide sequence design；Preferably, the lncRNA TCONS_00027980 transcript cores
Nucleotide sequence is SEQ ID NO.1.
9. inhibitor as claimed in claim 8, it is characterised in that the inhibitor is one kind or more in 6 kinds of siRNA
Kind；
1)siRNA-1：SEQ ID NO：6atgatgtgggatgtacaag
2)siRNA-2：SEQ ID NO：7tgcttgatgatgtaggtcc
3)siRNA-3：SEQ ID NO：8tcattgttgatttgtcagc
4)siRNA-4：SEQ ID NO：9agccatgatgtgggatgtac
5)siRNA-5：SEQ ID NO：10cctccttccttcatgtggac
6)siRNA-6：SEQ ID NO：11ccctcctcgtgtgattgcag.
10. a kind of preparation for being used to adjust hepatocyte growth, it is characterised in that the preparation is included as described in claim 8 or 9
Inhibitor.
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